Direct association of calponin with specific domains of PKC-alpha.
Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-alpha in the membrane. In the present study, we have determined the domain of PKC-alpha involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-alpha. Calponin was found to bind directly to the full-length PKC-alpha. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-alpha. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with His-tagged proteins of individual domains and different combinations of domains of PKC-alpha. Coimmunoprecipitation of calponin with His-tagged truncated forms of PKC-alpha showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-alpha and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-alpha and that the functional association of PKC-alpha with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells.
Somara
Sita
S
Division of Pediatrics-Gastroenterology, University of Michigan Medical Center, Ann Arbor, MI 48109-5656, USA.
Bitar
Khalil N
KN
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003106:Colon / Q000166:cytology / Q000187:drug effects; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000096985:Calponins
D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D051571:Protein Kinase C-alpha; D011993:Recombinant Fusion Proteins; D000096985:Calponins